The silkworm cell line BmN originated from ovarian was used in this study. BmN cells were maintained in our lab, culturing in TC-100 insect medium supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotic–antimycotic mixture at 27°C.

To determine the required concentration of rotenone, cell viability was measured using Cell Counting Kit-8 (CCK-8) (MedChemExpress, China). Briefly, BmN cells were subcultured in 24-well plates at a seeding density of 5 × 10⁴ cells per well and treated with rotenone (0.05, 0.5, 1, 10, or 50 μg/ml) for 2 days. Rotenone was diluted in dimethyl sulfoxide (DMSO). After treatment, 10 μl of CCK-8 solution was added to each well of the plates, and the plates were kept in the incubator at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader. Data were expressed as mean ± SD from six independent experiments.

To comprehensively evaluate the impact of rotenone on BmN cells, we categorized the cells into distinct groups for detailed investigation. Group I, designated as the Control group, involved the incubation of BmN cells in a medium supplemented with DMSO for a period of 2 days. Meanwhile, in Group II, labeled as the Rotenone group, BmN cells were exposed to a medium containing freshly diluted rotenone at a concentration of 1 μg/ml for a duration of 2 days. It is worth noting that rotenone was meticulously prepared and diluted in DMSO to ensure optimal experimental conditions.

Detection of the mitochondrial membrane potential (MMP) was performed using a HCS Mitochondrial Health Kit (Molecular Probes™, Invitrogen) according to the manufacturer’s instructions. After drug treatment, 50 μl of cell staining solution containing 10.5 μl of the MitoHealth stain solution and 2.1 μl of Image-iT^® DEAD Green™ viability stain was added to each well of the plates. After 30 min of incubation at 27°C, the medium was removed and the cells were incubated with 100 μl of counterstain/fixation solution containing 6 μl of Hoechst 33342 for 15 min. Finally, the counterstain/fixation solution was removed and the wells were rinsed with 100 μl of PBS. A fluorescent microscope was used to scan the plates.

To study the formation of mitolysosome, mitochondria and lysosomes in BmN cells were labeled using MitoTracker^® Red CMXRos (Thermo Fisher Scientific) and LysoTracker^® Green DND-26 (Thermo Fisher Scientific), respectively, with reference to the manufacturer’s instructions. After drug treatment, the medium was removed from the plates and pre-warmed staining solution containing MitoTracker^® Red probe and LysoTracker^® Green probe was added, followed by 3 h of incubation. Co-localization of mitochondria and lysosomes were observed by a fluorescent microscope (LSCM system, LSM800, Zeiss, Germany) in the dark.

Total RNA was extracted from cultured cells using TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. Afterward, mRNA was enriched by Oligo(dT) beads, fragmented into short fragments using a fragmentation buffer, and reversely transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments were subjected to end repair, a base addition, and ligation of Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1.0X), followed by polymerase chain reaction (PCR) amplified. The resulting cDNA library was sequenced on the Illumina Novaseq6000.

After sequencing, raw reads were preprocessed using fastp (v.0.23.4) by removing adaptor sequences and filtering low-quality reads (Chen, 2023). FastQC (v.0.12.1) was used for quality control before and after preprocessing (Patel and Jain, 2012). The trimmed and filtered high-quality clean reads were aligned to the B. mori genome (GCF_014905235.1) by HISAT2 (v.2.2.1) with default parameters (Kim et al., 2019), and the gene expression levels were estimated using featureCounts (v.2.0.3) (Liao Y. et al., 2014). Subsequently, a differentially expressed analysis of silkworms involved in rotenone and DMSO was conducted via DESeq2 (v.1.38.3) with default parameters (Love et al., 2014). To address the high rate of false positives resulting from multiple testing, the Benjamini–Hochberg method was used to adjust the p-value. Genes with adjusted p-values < 0.05 and an absolute log2(Fold Change) > 1 were recognized as differentially expressed genes (DEGs). These DEGs were subjected to the Gene Ontology (GO) function analysis and KEGG pathway enrichment using clusterProfiler (v.4.6.2) with a Benjamini–Hochberg corrected p-value < 0.05 (Wu et al., 2021). The analysis results were presented in the form of bubble and hierarchical bar charts.

The B. mori (strain: 306) was obtained from the Institute of Life Sciences, Jiangsu University. The silkworm larvae were reared with fresh mulberry leaves under the condition of 25 ± 2°C and 12 h light/dark cycle. On the first day of fifth instar, 90 larvae were randomly divided into three groups: Control group, DMSO group, and Rotenone group (n = 30/condition). Drops of water, DMSO, and 50 μg/ml rotenone were applied to the heads of the silkworms in Control group, DMSO group, and Rotenone group, respectively. After 24 h, 4 larvae were randomly selected from each group to determine the motor function. Another treatment was performed on 15 selected silkworms from each group, following the same procedure as the first round. After 24 h, four individuals were selected from each group for motion testing. Figure 1 showed the method used to assess the motor function of silkworms. Each silkworm was placed on a strip of cardboard and the time it took them to move from their starting point to the mulberry leaves was recorded. In addition, six individuals were selected from each of DMSO group and Rotenone group for brain tissue collection. The brain tissues were washed twice with sterile PBS, packed into 1.5 ml EP tubes, frozen with liquid nitrogen, and stored in a refrigerator at −80°C.

Total RNA was isolated with UNlQ-10 Column TRIzol Total RNA Isolation Kit (Sangon Biotechnology, Shanghai, China) and reversely transcribed into cDNA using HiScript III RT SuperMix with gDNA wiper (Vazyme, Nanjing, China). Gene expression levels were quantified by an ABI 7,500 Sequence Detector (Applied Biosystem, Carlsbad, CA, USA) with the AceQ qPCR SYBR Green Master Mix (Vazyme Biotechnology, Nanjing, China). Primer sequences synthesized by GENEWIZ were shown in Table 1. Reactions were carried out with 2.8 μl reverse transcribed RNA and gene-specific primers in a total volume of 20 μl. PCR conditions consisted of an initial incubation step of 2 min at 50°C and a heat activation step of 10 min at 95°C. This was followed by 40 cycles of denaturation at 95°C for 10 s and annealing and extension at 60°C for 30 s.

The viability of BmN cells treated with increasing concentrations of rotenone was examined by CCK-8 assay and the results indicated that exposure to rotenone caused a dose-dependent decrease in cell viability. As shown in Figure 2, increasing the concentration of rotenone from 0.05 to 1 μg/ml did not result in a significant change in cell viability. However, when the concentration was further increased to 10 μg/ml, a significant decrease in cell viability was observed in comparison with the cells treated with 0.05, 0.5, and 1 μg/ml rotenone. After 48-h rotenone treatment, the cell viability was 39.72% in the 10 μg/ml rotenone-exposed group, while only 28.83% was left in the highest concentration group (50 μg/ml), implying that rotenone can exert an antiproliferative effect. To further investigate the adverse effects of rotenone on mitochondrial function and mitophagy, BmN cells were treated with 1 μg/ml rotenone.

The cytotoxic effect of rotenone is known as the inhibition of mitochondrial complex I, which may lead to mitochondrial dysfunction and damage (Pravdic et al., 2012; Basit et al., 2017; Heinz et al., 2017; Morales-Martinez et al., 2022). Since the MMP is a key indicator for evaluating mitochondrial function, we measured the changes in MMP in BmN cells treated with DMSO or rotenone using HCS Mitochondrial Health Kit (Sakamuru et al., 2016). As shown in Figure 3A, the red fluorescence of the MitoHealth stain in the Rotenone group was significantly lower than that of the Control group after 6, 12, and 24 h of rotenone treatment, suggesting a decrease in MMP in rotenone-treated BmN cells (Figure 3B). Mitochondrial dysfunction is an essential precursor event that triggers mitochondrial autophagy (Lu et al., 2023). To maintain mitochondrial as well as cellular homeostasis, cells use the mechanism of mitochondrial autophagy to deliver damaged mitochondria to lysosomes by autophagosomes for degradation (Xu et al., 2020). Therefore, to investigate the effect of rotenone on mitophagy levels, we labeled mitochondria and lysosomes with MitoTracker^® Red probes and LysoTracker^® Green probes, respectively. The formation of mitochondria-containing autolysosomes was indicated by yellow fluorescence. As shown in Figure 4A, the appearance of mitochondria-containing autolysosomes was clearly observed in cells after 12 h of rotenone treatment, whereas it was not observed in cells following 12 h of DMSO treatment. Furthermore, we employed Pearson’s correlation coefficient to estimate the colocalization of mitochondria and autolysosomes. Pearson’s R value between MitoTracker^® Red and LysoTracker^® Green increased from 0.01 in DMSO-treated cells to 0.42 in rotenone-treated cells (Figure 4B). Together, the images and correlation analyses consistently indicated increased mitophagy levels after rotenone treatment.

In order to investigate the effect of rotenone on gene expression in BmN cells, high-throughput transcriptome sequencing was performed for the DMSO-treated group (Control group) and the rotenone-treated group (Rotenone group), with five replicates for each group. After preprocessing, 40,493,214, 39,872,296, 37,184,136, 42,461,534, and 40,465,356 clean reads were obtained from the Control group and 37,397,810, 39,760,342, 38,444,916, 42,751,288, and 41,371,804 clean reads were obtained from the Rotenone group. For the Control group, the GC content and Q30 percentage of the clean data ranged from 46.59% to 47.67% and from 92.75% to 93.65%, and a mean of 76.58% of clean reads was mapped to the B. mori genome. Besides, for the Rotenone group, the GC content and Q30 percentage of the clean data ranged from 41.36% to 48.50% and from 92.52% to 93.14%, and a mean of 75.45% of clean reads was mapped to the B. mori genome (Table 2). The results of principal component analysis (PCA) and Pearson correlation analysis demonstrated that all 10 samples were clearly divided into two clusters, and that there were significant differences between these two clusters (Figure 5).

Differentially expressed analysis was performed using the R package DESeq2 to identify DEGs between the Rotenone group and the Control group to investigate the impact of rotenone on gene expression levels. A total of 1,454 genes passed the fold change (FC) and corrected p-value filters (absolute log₂FC > log₂1.5, corrected p-value < 0.05), including 877 significantly upregulated and 577 downregulated in the Rotenone group compared to the Control group, as represented in a volcano plot (Figure 6).

To elucidate the potential functions and related biological processes of the 1,454 DEGs, we performed the GO function analysis and KEGG pathway enrichment on these genes using the R package clusterProfiler. The results with corrected p-values < 0.05 were considered statistically significant.

The number of GO terms in the biological process (BP), cellular component (CC), and molecular function (MF) categories obtained from the GO enrichment analysis for upregulated DEGs were 51, 2, and 37, respectively. In downregulated DEGs, the number of GO terms in the BP, CC, and MF categories were 2, 3, and 4, respectively. We chose the primary BP, CC, and MF terms from each analysis result for visualization (Figures 7A, C). The enrichment of “mitochondrion,” “mitochondrial membrane,” “mitochondrial inner membrane,” “mitochondrial protein-containing complex,” “inner mitochondrial membrane protein complex,” and “mitochondrial envelope” suggested that rotenone may impact mitochondria in BmN cells.

Furthermore, KEGG pathway enrichment analysis showed that a total of 7 KEGG pathways were significantly enriched. The upregulated DEGs were mainly enriched in six pathways, including “Ribosome,” “Protein processing in endoplasmic reticulum,” “Proteasome,” “Oxidative phosphorylation,” “mitophagy,” and “Lysosome,” whereas the downregulated DEGs were mainly enriched in eight pathways: “Toll and Imd signaling pathway,” “Peroxisome,” “Notch signaling pathway,” “Mitophagy,” “MAPK signaling pathway,” “Carbon metabolism,” “Autophagy,” and “Apoptosis” (Figures 7B, D). These results indicated that rotenone’s toxicity may be related to its effects on ATP synthesis, removal of dysfunctional mitochondria or other intracellular structures, and cell death.

PTEN induced putative kinase 1 (PINK1)/Parkin pathway may be involved in the mitophagy induced by rotenone. Hence, we analyzed the expression levels of genes that encode the primary components in the PINK1/Parkin-mediated mitophagy. The transcriptome analysis and quantitative real-time PCR (qRT-PCR) results showed that in Rotenone groups, the expression of RpS27a, LOC692779, and ATG8 was significantly elevated, while there was no difference in USP30 expression (Figures 6, 8, 9). Furthermore, the genes MFN2, Parkin, USP8, p62, and TBK1 were not identified as DEGs. However, the results of RT-qPCR showed a significant increase in the mRNA levels of MFN2, Parkin, p62, and TBK1 in Rotenone groups (Figures 6, 8, 9). In addition, the differentially expressed analysis revealed significant downregulation of AMBRA1 and upregulation of ATF4 in Rotenone groups, while RT-qPCR results were the opposite (Figures 6, 8, 9). These findings indicated that rotenone may induce PINK1/Parkin-mediated mitophagy.

To study the effect of rotenone on the movement of silkworms, we placed the silkworms treated with water, DMSO, or rotenone at starting points on cardboard boards with mulberry leaves at the opposite ends. We then measured the time it took the silkworms to crawl from the starting point to the mulberry leaves to assess their mobility. Twenty-four hours after the first treatment, the silkworms with rotenone dropped on their heads moved significantly faster than those with water or DMSO, which may indicated that the rotenone treatment had activated their survival instinct, causing them to crawl toward the mulberry leaf (Figure 10A). However, 24 h after the second treatment, the movement speed of the rotenone-treated silkworms was significantly slower than that of the water- or DMSO-treated silkworms, suggesting that rotenone may induce motor dysfunction in the silkworms (Figure 10A). Dopamine is a neurotransmitter that regulates motion (Bertoldi, 2014). To investigate whether rotenone also affected dopamine synthesis, we analyzed the expression of Tyrosine hydroxylase (TH). As shown in Figure 10B, the level of TH expression was significantly downregulated in Rotenone groups. Since TH is the rate-limiting enzyme in dopamine synthesis, suppression of TH expression may disturb dopamine synthesis (Daubner et al., 2011). This could partly explain why rotenone impaired the locomotion of the silkworms. Together, these results suggested that the phenotypes of the silkworms treated with rotenone shared similarities with some symptoms of PD in humans. This indicated that rotenone showed promise for use in establishing a silkworm-based PD model.